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Abstract

Background

Repetitive Transcranial Magnetic Stimulation (rTMS)/ Deep-brain Magnetic Stimulation
(DMS) is an effective therapy for various neuropsychiatric disorders including major
depression disorder. The molecular and cellular mechanisms underlying the impacts
of rTMS/DMS on the brain are not yet fully understood.

Results

Here we studied the effects of deep-brain magnetic stimulation to brain on the molecular
and cellular level. We examined the adult hippocampal neurogenesis and hippocampal
synaptic plasticity of rodent under stress conditions with deep-brain magnetic stimulation
treatment. We found that DMS promotes adult hippocampal neurogenesis significantly
and facilitates the development of adult new-born neurons. Remarkably, DMS exerts
anti-depression effects in the learned helplessness mouse model and rescues hippocampal
long-term plasticity impaired by restraint stress in rats. Moreover, DMS alleviates
the stress response in a mouse model for Rett syndrome and prolongs the life span
of these animals dramatically.

Keywords:

Background

Transcranial magnetic stimulation (TMS) is a non-invasive approach of brain stimulation,
which utilizes an insulated coil placed over the scalp and induces neural activity
within the brain [1-3]. TMS is commonly applied in single, paired or repetitive trains. Repetitive TMS (rTMS)
has been proven to modulate motor skills and cognitive function in healthy subjects
and exhibits therapeutic effects for patients with neurological and psychiatric disorders
[4,5]. Daily prefrontal TMS was approved by Food and Drug Administration (FDA) in 2008
for the treatment of patients with major depressive disorder. Recently, deep-brain
magnetic stimulation (DMS) with a modified rTMS protocol has been developed and demonstrated
to be effective for Parkinson’s disease and neuropsychiatric disorders, including
depression [6,7]. Despite the indubitable contribution of rTMS/DMS to cognitive and motor functions
of nervous system, the molecular and cellular mechanisms underlying the anti-depression
effects of rTMS/DMS remain largely unknown. The prevailing hypothesis is that rTMS
may stimulate neural activity in certain brain regions by modulating the balance between
excitatory and inhibitory neurons [8-12].

Depression is a leading cause of psychiatric disability worldwide. During the past
decade, the adult neurogenesis hypothesis of depression has been widely accepted,
which postulated that the decline in adult hippocampal neurogenesis contributes to
the pathophysiology of depression, while the biogenesis of adult new-born neurons
in the dentate gyrus (DG) of hippocampus is required for the beneficial effects of
antidepressant treatment [13-15]. In non-human primates, stress led to a decrease in adult hippocampal neurogenesis,
which was rescued by antidepressant treatment [16]. Chronic rTMS treatment exhibited robust anti-depression effects in animal models
and might enhance adult neurogenesis under chronic stress [17,18].

Here we show that DMS increases the proliferation of hippocampal neural progenitor
cells in the adult brain, promotes the dendritic complexity of new-born neurons and
enhances the neuronal activity in hippocampus, indicated by the up-regulation of activity-dependent
genes. Moreover, administration of DMS not only alleviates the depression and anxiety-associated
behaviors of a mouse model for Rett syndrome, but also strikingly prolongs the lifespan
of these animals.

Results and discussion

DMS promotes adult hippocampal neurogenesis

To examine the effects of DMS on adult hippocampal neurogenesis in wild-type animals,
we applied the DMS protocol to 6 weeks old wild-type mice 20 minutes daily. The schematic
illustration of the DMS device is shown in (Additional file 1: Figure S1A). Two magnetic stimulating coils are symmetrically placed on the both
sides of the mouse cage. The stimulus device can be discharged through two coils to
produce a time-varying pulsed magnetic field. The first stimulating procedure (Program1)
is shown in Figure 1A. In this strategy, the magnetic flux density is linear gradient. The magnetic pulse
is bipolar, as shown in Additional file 1: Figure S1C,E. The second stimulating paradigm (Program 5) is shown in Figure 1B. Different with Program1, the magnetic flux density of Program 5 is uniform alternating
linear gradient, both magnetic fields are illustrated in Additional file 1: Figure S1B-E. First, we examined whether DMS stimulation would have global effects
on the general health of experimental animals. We measured the body weights of mice
receiving either Program 1 (P1) or Program 5 (P5) treatment daily for 3 weeks and
found that P1 or P5 treatment didn’t affect the body weight changes comparing to naïve
mice (Figure 1C).

Figure 1.Schematic illustration of the new DMS paradigm. (A) Illustration of Uniform and Linear magnetic fields generated by this equipment. Composition
of Program 1 (A) and Program 5 (B). (C) Body weight changes of mice under control, Program1, and Program5 treatment daily
for 3 weeks. (n = 6 for each condition).

To investigate the role of DMS in the proliferation of neural stem cells, we applied
a short-term bromodeoxyuridine (BrdU) labeling strategy to monitor postnatal hippocampal
neurogenesis in the mice subjected to DMS with P1 or P5, respectively. For the thymidine
analog incorporation assay, BrdU was administered into adult mice 4 times with 2-hour
interval by intraperitoneal injection after 4 or 7 days treatment of DMS (Figure 2A). The treated animals were sacrificed 2 hours after the last time of BrdU injection.
The staining of BrdU labels the new-born neurons in the hippocampus. The results showed
a dramatic increase in the number of active proliferating neural progenitor cells
in the subgranular zone of DG after administration of DMS with P1 for 7 days rather
than 4 days (Figure 2B,C). Intriguingly, stimulation with P5 significantly elevated the production of new-born
neurons in the DG as early as 4 days after treatment, suggesting that different DMS
paradigms may generate various biological effects for adult neurogenesis (Figure 2D,E).

To further confirm whether DMS treatment enhances proliferation of neural progenitors,
we further performed immunostaining using antibody against Ki67, a cell proliferating
marker on these samples. We found that the number of Ki67- positive cells was significantly
increased in DG area after P5 treatment for 7 days, indicating that DMS treatment
promotes neural progenitor proliferation (Figure 3A,B). To validate that the BrdU-labeled cells are proliferating progenitors, we performed
co-immunostaining for both BrdU and Ki67 and found 100% of colocalization between
the two markers. (Additional file 2: Figure S2A-D). Furthermore, to reveal the cellular identity of BrdU-positive cell
we performed immunostaining using antibodies against more neural progenitor markers
including radial glial-like cell (RGL) marker glial fibrillary acidic protein (GFAP),
immature neuron marker doublecortin (DCX), and intermediate progenitor (IPC) marker
T-box brain 2 (Tbr2). We found that cells labeled by short-term BrdU pulse are during
actively proliferating process and show 63% (control) versus 67% (DMS) co-localization
with Tbr2, 21% (control) versus 16% (DMS) with GFAP, and 14% (control) versus 15%
(DMS) with DCX (Additional file 2: Figure S2E-H and Additional file 3: Figure S3A-H). Taken together, our results indicated that DMS promotes not only
the self- renewal of RGLs but also their differentiation into IPCs and late neuroblasts.

Next, we examined the effect of DMS treatment on adult hippocampal neurogenesis by
in aged animals, in which the rate of adult neurogenesis remains in a much lower level
than adult mice. The observation that aging has a negative effect on the proliferation
of neural stem cells [19] has called up efforts to boost neurogenesis in senescent animals, which will have
beneficial effects for age-related cognitive decline. To study whether DMS improves
the reduced neurogenesis in senescent mice, we applied daily DMS in 9-month old mice
for 7 days and examined the neurogenesis by BrdU incorporation assay. Our results
revealed that the population of proliferating neural progenitors was significantly
enhanced after DMS treatment in P5, while DMS with P1 had a slight increase but not
statistically significant effects on hippocampal neurogenesis (Figure 3C,D). Thus, we demonstrated that DMS with patterned stimulation protocols promoted
the proliferation of hippocampal neural progenitor cells in both adult and senescent
animals.

DMS facilitates the development of new-born neurons

Adult hippocampal neurogenesis largely recapitulates the process of neural development
in embryonic stages. After birth, new-born neurons in the DG migrate into the granule
cells layer, extend dendrites toward the molecular layer, project axons through the
hilus toward the CA3 and integrate into the existing circuitry [20,21].

To address the effects of DMS on the maturation of new-born neurons, we labeled these
neurons by stereotactic injection of retrovirus into the DG of young mice, followed
by the 2-week daily treatment of DMS with distinct programs (Figure 4A) [22]. Retroviral infection of proliferating neural progenitor cells with the introduction
of green fluorescent protein (GFP) allows the sparse labeling of new-born neurons
and the elaborate observation of their morphological complexity. The morphology of
GFP labeled new-born neurons was examined with confocal microscopy at 2 weeks post-injection
(Figure 4B). The results showed that there was a significant increase in both the number of
the tips and total dendritic length after DMS treatment with either P1 or P5, when
compared to the control GFP labeled neurons (Figure 4C-E). We further applied DMS with Program 5 up to 4 weeks to mice which received retrovirus
injection ahead of stimulation and analyzed the dendritic morphogenesis of new-born
neurons (Figure 4F). Consistently, the results revealed that DMS robustly facilitated the dendritic
growth and branching of neurons (Figure 4G-I).

In order to examine whether DMS may affect the development of mature neurons, we performed
Golgi staining on the brain sections from control and DMS-treated animals (Additional
file 4: Figure S4A,B). We measured the spine density of DG granule neurons after DMS for
2 weeks and found that DMS has no effects on spine density of fully mature neurons
(Additional file 4: Figure S4C,D).

Collectively, these evidences demonstrated that DMS treatment not only enhances the
proliferation of adult neural progenitor cells in DG, but also facilitates the development
and maturation of new-born neurons, suggesting that new-born neurons induced by DMS
are able to incorporate into hippocampal neural circuitry and contribute to neuronal
plasticity of the central nervous system.

DMS stimulates gene expression in hippocampus in vivo

To further explore the consequences at the molecular level caused by DMS treatment,
we performed immunostaining and quantitative PCR to examine the change of gene expression
in the hippocampus in vivo. Neural activity induces the modification of synaptic proteins, promotes local protein
synthesis within dendrites and activates gene transcription in neurons. Therefore,
we compared the expression level of several activity-dependent genes in the hippocampus
between control groups and DMS-treated groups. The immediate early genes including
c-fos, egr1 and arc are commonly used as markers for measuring neuronal activity in the brain. We first
examined the expression of immediate early gene c-fos, in the DG after daily DMS with P5 paradigm for 4 days by immunohistochemistry. The
results of immunostaining showed that the population of c-fos-positive neurons was
significantly increased in DMS-treated animals (Figure 5A,B). Additionally, the Fgf1b gene is a brain-specific expressing gene encoding an
important neurogenic niche factor in the adult hippocampus, and is induced by electroconvulsive
stimulation [23]. We collected the hippocampal samples from animals with or without DMS treatment,
further examined the mRNA levels of fgf1b in the DG by quantitative PCR. We found that the expression level of fgf1b was remarkably elevated after DMS (Figure 5C). Furthermore, we examined the protein level of Fgf1 in hippocampus with DMS treatment
for 4 days. Consistently, we found that DMS treatment for 4 days significantly lead
to an increase of protein level of Fgf1 in hippocampus (Figure 5D,E). These results suggest that DMS stimulation increases neural activity in the
hippocampus, induces the expression of activity-dependent gene and leads to the release
of neurogenic niche factors, which thereby promotes neurogenesis and plasticity in
the adult brain.

DMS exerts anti-depression effects on rodent model

It is postulated that the defects in adult hippocampus neurogenesis is strongly associated
with major depressive disorder. Exercise may alleviate depression symptoms via enhancing
neurogenesis in animals [24]. The rTMS has been an effective therapeutic tool for the treatment of several neuropsychiatric
disorders in human patients, as well as rats with chronic unpredicted mild stress
(CUMS) [25]. The evidence that application of DMS to wild type animals increases adult neurogenesis
prompted us to investigate whether the newly-developed DMS protocol has anti-depressive
effects in the rodent model. We applied learned helpless animal paradigm to model
some aspects of depression in rodents [26]. Following 2 days of unpredicted electric foot-shocks, we successfully induce the
depression phenotype in mice assessed by forced swimming test, in which mice with
foot-shocks appeared to have increased immobility time compared to naïve animals (Figure 6A,B, Additional file 5: Figure S5A-E). After treating mice with continuous 5-day DMS treatment with P5,
we found that the immobility time of depressed mice induced by foot-shock was fully
rescued to normal level by the DMS treatment (Figure 6B).

The pathophysiology of major depressive disorder has been characterized by alternations
of molecular markers. Whole-genome expression profiling revealed that mitogen-activated
protein kinase phosphatase-1 (MKP-1) was dysregulated in the hippocampal tissues from
patients with depression, suggesting that MKP-1 serves as a molecular marker for depression
[27]. Consistently, the expression level of MKP-1 was significantly up-regulated in the
hippocampus of mouse model with depression phenotypes (Figure 6C). Importantly, the dys-regulated expression of MKP-1 in depressed mice was completely
restored by the DMS treatment (Figure 6C).

Next we investigated whether increased adult hippocampal neurogenesis induced by DMS
plays a critical role in alleviation of depressive behavior by elimination of neurogenesis
using gamma irradiation (IR). After applying IR after foot shock, we found that IR
abolished the effect of DMS on alleviation of depressive behaviors (Figure 6D,E). Meantime, we found that IR itself also leads to a dramatic decrease of hippocampal
adult neurogenesis (Additional file 6: Figure S6A-C).

Furthermore, mood disorder induced by acute stress impairs long-term potentiation
(LTP) in the neural circuitry of hippocampus [28-31]. The effects on synaptic plasticity occur following a plethora of stressors including
administration of shock, exposure to a predator or a novel environment [32]. We used chronic restraint stress paradigm to induce depression in rats and recorded
LTP in hippocampus by in vivo electrophysiological recording. Indeed, LTP was found to be impaired in rats undergoing
restraint stress (Figure 6F). Notably, DMS treatment for 7 days largely rescued the deficit LTP induction in
depressed rats (Figure 6F,G). These behavioral, molecular and electrophysiological evidences strongly support
that short-term DMS treatment is effective for alleviating depression-associated phenotypes
in animal models.

Finally we would like to further examine whether DMS could alleviate the anxiety-related
behaviors in other disease models. Rett syndrome is a severe neural developmental
disorder, primarily caused by loss-of-function mutations of gene MECP2 (Methyl-CpG-binding protein 2) [33-35]. The mouse carrying a truncated form of MeCP2 protein (mecp2308/y) mimics severe phenotypes of Rett syndrome patients, including elevated anxiety and
stress responses [36]. Thus we asked whether DMS could help to relieve the anxiety-associated phenotypes
in mice carrying mecp2308/y mutation.

We first examine stress responses in mecp2308/y mouse and its wild type littermates, using light–dark transition paradigm. We found
that mice carrying mecp2308/y mutant appeared to spend much less time in light compartment and thus to have increased
anxiety levels (Figure 7A,B). Interestingly, chronic daily DMS treatment for 5 months restored elevated anxiety
levels of mecp2308/y mice to normal level (Figure 7A,B). Thus, DMS treatment appears to have significant effects to regulate the anxiety
levels of animals for disease models.

Lastly, we asked whether DMS treatment would make positive impacts to the general
health of the mouse model of Rett syndrome. Lack of MeCP2 in mouse would lead to significant
shortage of lifespan [37,38]. We acquired nervous system specific mecp2 knockout mice by crossing mice carrying neuronal specific Cre transgene nestin-cre with mecp2 floxed allele- mecp2flox/y. We found that loss of MeCP2 in the nervous system would lead to substantial shortage
of lifespan down to around 130 days or so (Figure 7C). Surprisingly, mice with nestin-cre, mecp2flox/y received daily DMS with either P1 or P5 appeared to have enormously extended lifespan
(Figure 7C), indicating that long-term treatment of DMS indeed exerts positive effects on the
general health to mouse model of Rett syndrome.

Discussion

The growing interest in noninvasive brain stimulation generated by TMS led to its
widespread application to treat various neurological and psychiatric disorders including
major depression and Parkinson’s disease. The NeuroStar TMS Therapy system (Neuronetics,
Inc., Malvern, PA, USA) received FDA clearance for the treatment of adult patients
with intractable depression in 2008. It is assumed that magnetic stimulation makes
use of electromagnetic principles to alter neural activity non-invasively, and induces
focal as well as network effects in the brain. However, how rTMS changes the cellular
behavior and functional connectivity remains enigmatic.

Here we developed a novel rTMS paradigm, referred as DMS, to help the learned helpless
animals recuperate from stress and depression. The short-term application of DMS to
depressed mice not only improved their stress-related behavior but also amazingly
reversed the pathophysiology of major depression, indicated by the alteration of molecular
marker and neuronal plasticity. As the neurogenic hypothesis of depression gains momentum
over the last decade, more and more evidence confirmed that the waning and waxing
of neurogenesis in the hippocampus are important causal factors in the precipitation
of and recovery from depression, respectively. Our study provided several lines of
evidences to prove that DMS treatment increases adult neurogenesis and new-born neuron
maturation in vivo. First, short-term DMS administration rapidly induces the proliferation
of adult neural progenitors in the subgranular zone of DG. The enhanced neurogenesis
by DMS treatment occurs in both adult and senescent animals. Second, the application
of DMS to adult animals promotes the dendritic development new-born granule neurons,
suggesting the elevated incorporation of new-born neurons into existing hippocampal
circuitry might be achieved by magnetic stimulation. Finally, DMS treatment is shown
to up-regulate the neural activity, which is beneficial to increase the production
and release of neurogenic niche factors such as FGF1b. Thus, our results support the
notion that DMS is an efficient therapeutic treatment for major depression disorders
and the antidepressant effects of DMS may rely on the elevation of hippocampal adult
neurogenesis and the modulation of hippocampal synaptic plasticity.

Neural activity and experience, presumably acting on this local niche, regulate multiple
stages of adult neurogenesis, from neural progenitor proliferation to newborn neuron
maturation, synaptic integration and survival. Our study shows a new non-invasive
way to enhance adult hippocampal neurogenesis probably by changing the network activity
of hippocampus and inducing a beneficial neurogenic niche in the subgranular zone.
Repetitive electrical stimulation in adult hippocampal slices induces NMDA receptor-dependent
LTP and high frequency stimulation results in persistent increase in synaptic strength
[39]. Studies using theta-burst stimulation protocols provide solid evidence linking human
rTMS with LTP-like plasticity [40,41]. The DMS protocol we applied exerts high frequency oscillated stimulation in the
brain of animal models and rescues the deficient LTP impaired by restraint stress
in rats. On the other hand, LTP induction in the anesthetized rats increases neurogenesis
in the dentate gyrus and FGFR activation by the neural cell adhesion molecule promotes
neural progenitor proliferation by enhancing LTP [42,43]. Therefore, the study suggests that DMS administration may promote adult neurogenesis
by inducing high frequency theta-burst stimulation and LTP in the hippocampus. Further
study will be performed to explore the alteration of field potential by DMS treatment.
Moreover, the finding that DMS alleviates stress response and prolongs the life expectancy
of mouse model for Rett syndrome is particularly intriguing. Many trials have been
carried out to examine whether drug delivery or genetic manipulation help to alleviate
the impaired behavior and severe pathophysiology of Rett syndrome mouse model [44-46]. Although gene delivery of Mecp2 seems to completely rescue various symptoms in animal models of disease, the efficacy
and safety issues of gene delivery remain controversial. Here our results suggest
a modified DMS paradigm which may relieve the stress-related symptoms, as well as
improve the life quality of Rett syndrome patients. Further study of how DMS treatment
may contribute the neural plasticity of the central nervous system under disorder
conditions are very critical for deepen our understanding of therapeutic efforts towards
the cure of neurodevelopmental disorders and neuropsychiatric disorders. In order
to develop easier and more effective therapy for patients with neurodevelopmental
disorders and neuropsychiatric disorders, we need to better understand how DMS treatment
contributes to the neural plasticity of central nervous system under pathological
conditions.

Conclusion

Taken together, we report that a new DMS paradigm rapidly induces adult hippocampal
neurogenesis and promotes development of DG new-born neurons. Our study provides a
new non-invasive way to enhance adult hippocampal neurogenesis within a short period
of time and make it possible to study the role of adult hippocampal neurogenesis in
a gain-of-function manner. More importantly, we provided evidences that this new DMS
paradigm efficiently rescues behavioral phenotypes and gene expression profiles in
learned helplessness mouse model, as well as restoring hippocampal LTP impaired by
restraint stress in rats. These results support the notion that DMS is an efficient
therapeutic treatment for major depression disorders and strongly suggest that the
antidepressant effect of DMS on depression disorders may rely on the improvement of
hippocampal adult neurogenesis and the correction of hippocampal synaptic plasticity.

Methods

Animals

Animals were group-housed with free access to water and food in the established animal
houses, with a 12 hours light/dark cycle and a thermo-regulated environment. The use
and care of animals complied with the guideline of the Biomedical Research Ethics
Committee at the Shanghai Institutes for Biological Science, CAS. Adult (6–7 weeks
old) male C57BL/6 mice (SLAC Laboratory Animal) were used in all experiments except
electrophysiology trials which were performed on male Sprague Dawley rats (Animal
House center, Kunming General Hospital, Kunming), weighing 220–250 g. mecp2308/y mice (005439) and nestin-cre mice (003771) were purchased from Jackson lab; floxed
mecp2 (011918) were purchased from MMRRC at UC Davis.

DMS treatment

For DMS treatment, mice with their cage were placed in DMS machine. Metal parts of
cages were removed prior to DMS treatment. The applied program was described in Supplementary
Figures. Briefly, twenty minutes successive trains of DMS were administered daily
for different consecutive days depending on the purpose of experiments.14 days DMS
were administered on retrovirus injected mice and 4 or 7 days DMS on Brdu labeled
mice while 5 days DMS on acute induced depression mice. The control group conditions
were identical to their DMS group but received sham stimulation. For electrophysiology
experiments, control group received no treatment, Restraint stress (RS) group were
restrained in the fixing cage 20 min each day for 7 days, RS + DMS group received
20 min DMS treatment in the fixing cage for 7 days. Electrophysiology studies were
carried out 0.5 h later after the last restraint stress or DMS treatment.

In vivo electrophysiology

Experiments were carried out on rats anesthetized with pentobarbital (60 mg/kg, i.p.),
and core temperature was maintained at 37 ± 0.5°C. Recordings of field excitatory
postsynaptic potentials (EPSPs) were made from the CA1 stratum radiatum of the hippocampus
in response to ipsilateral stimulation of the Schaffer collateral/commissural pathway
using techniques described previously [28,47]. Recording and stimulating electrodes were made by gluing together a pair of twisted
Teflon-coated 90% platinum and 10% iridium wires (50 μm inner diameter, 75 μm outer
diameter; World Precision Instruments, Sarasota, FL). The recording electrode was
inserted 3.8 mm posterior to bregma and 2.8 mm right of the midline, and the stimulating
electrode was inserted 4.8 mm posterior to bregma and 3.8 mm right of the midline.
The optimal depth of the wire electrodes in the stratum radiatum of the CA1 area of
the dorsal hippocampus was determined by electrophysiological criteria. Test EPSPs
were evoked at a frequency of 0.033 Hz and at a stimulus intensity adjusted to give
an EPSP amplitude 50% maximum response. The high frequency stimulation (HFS) protocol
for inducing LTP consisted of 10 stimulus trains of 20 pulses at 200 Hz, with 2 s
intertrain intervals. EPSP amplitude was expressed as mean ± S.E.M% of the baseline
EPSP amplitude recorded over a 40-min baseline period, and amplitudes in the last
10-min of recording were averaged in one animal and then across animals to give a
value for the group.

Preparation and stereotaxic injection of retrovirus

We thank Drs. Hongjun Song (Johns Hopkins University School of Medicine) and Zhengang
Yang (Institutes of Brain Science, Fudan University) for providing retroviral constructs
and supernatant containing retrovirus. Cell debris were removed from supernatant through
0.22 μm filter and the filtered supernatant was centrifuged at 65000 g at 4°Cfor 2 h.
Then supernatant was removed and 50 μl PBS was used to re-suspend virus. We seed 293 T
to determine the titer of virus. High titer virus of 108vg/ml was necessary to label new-born neurons in the adult DG.

For stereotaxic injection, it is critical to locate the exact hippocampus neurogenic
area. The mouse was mounted onto the stereotaxic frame, orienting the head straight
in terms of anterior-posterior axis and horizontally. Then we shaved a small area
by a trimmer, cut the skin over the scull, cleaned up the blood in the wound.

The needle was firstly moved to the bregma and then moved posteriorly 2.0 mm and laterally
1.5 mm to the injection position. A small hole was made on the scull using an electric
drill and the needle tip was moved down by 2.25 mm from the water level of the hole.
1 μl retrovirus solution was injected to one side.

For both Brdu and retrovirus stereotaxic injection statistics, each group (control,
program1 and program5) has 4–5 mice. Each mouse was collected 9–12 slices from dorsal
to ventral region of DG. And 1 or 2 intact neurons were chosen from each slice for
analysis. ImageJ software (http://rsbweb.nih.gov/ij/webcite) was used to trace the dendrites of new-born neuron. Then the dendritic length and
tips number were auto-analyzed.

Bromodeoxyuridine injection

Mice received four or seven days DMS treatment and Bronodeoxyuridine (Brdu) (50 mg/kg
in saline, every two hours for four times, Sigma, flu/Ald, B5002) was intraperitoneally
(i.p.) injected into the control or DMS group mice 12 hours after the last DMS treatment.
Mice were killed two hours later after the last i.p. injection.

Tissue preparation, immunostaining and imaging

Mice were transcardially perfused with 0.1 M cold PBS followed by 4% paraformaldehyde
(PFA) fixation. The brains were post-fixed for 12 hours in 4% PFA and dehydrated in
30% sugar solution for another 12 hours. Coronal sections of 40-μm thickness were
cut on the freezing microtome (LEICA CM1950) and stored in PBS.

For Brdu immunostaining, free-floating sections were incubated in 2 N HCl for 15 min
at 37 degree then neutralized in 0.1 M boric acid solution (pH 8.5) for 10 min and
washed by PBS for 3 times (each time for 5 minutes). Sections were incubated in mouse
anti-Brdu antibody (1:1500; MAB3510) at 4 degree overnight and washed again then incubated
in CF555 conjugated donkey anti-mouse secondary antibody (1:500; Biotum,20037) for
2.5 hours at room temperature.

Immunostaining sections were photographed in Z-series stacks using a Nikon A1 confocal
microscope. NIH Image J with NeuronJ plugin was used to count the Brdu-immunoreactive
cell number in the dentate gyrus and to quantify the area of the dentate gyrus, also
to analyze total dendritic length of GFP+ new-born neurons.

Statistical analysis

The results were expressed as Mean ± S.E.M. Statistical significance (P < 0.05) was assessed using the two-tailed Student’s t-test.

Learned helplessness paradigm

Mice were placed into the inescapable shock chamber. 360 scrambled foot shocks (0.6 mA)
with varying duration (1-3s) and interval-episodes (1-15s) were delivered over two
consecutive days. Control group did not receive foot shocks but were placed to the
shock chamber with equal time. On the first and second days, 8 hours after foot shocks,
mice were administered DMS. Then mice were administered DMS for another 3 days.

Forced swim test

Mice were placed individually in the testing cylinder (33 cm high × 10 cm in diameter)
containing water with 20 cm depth. The procedures were conducted as two minutes pretest
followed by four minutes test and both sessions were videotaped. Observers scored
the immobility (floating passively with slight movements) time in the test four minutes.

Gamma irradiation

Mice were anesthetized with 0.7% Pentobarbital sodium at 10ul/g, and exposed to cranial
irradiation at a field of 3.5×11mm above the hippocampus but other body parts were
protected with a 75 mm lead shield. Caesium-137 was used as radioactive source operated
by MDS Nordion GC-3000Elan. The dose rate was approximately 4 Gy per min. The procedure
lasted 5 min and 30 sec, delivering a total of 20 Gy in the center.

Light–dark transition

mecp2308/y mutant mice were carried out light–dark transition test after daily DMS stimulation
for 5 month at the age of 5–6 month. The chamber is divided into two unequal compartments
by an opaque shelter with a hole (of height 4 cm) at the floor level. The smaller
compartment (18cm×27cm×30cm) is painted black and covered by an opaque lid while the
larger compartment (27cm×27cm×30cm) is uncovered and is illuminated by ceiling room
lights. Firstly, mice were gently placed into the dark compartment. Transition between
the two compartments and time spent in the light compartment were automatically recorded
by the Ethovision videotracking system in a 10 min session.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

LX and ZQ conceived and supervised the project. YZ performed the majority of the work.
RRM and MT performed electrophysiology work on rat. ZFC, MH, ZRG, XL, DLD, participated
in early stage of this work including immunohistochemistry and daily DMS treatment
for animals. JW designed the DMS machine. XX and WHZ and CYL participated in testing
and data collection of DMS machine on animals. ZQ wrote the manuscript, with contributions
from other authors. All authors read and approved the final manuscript.

Acknowledgements

We thank Dr. Bai Lu for his constructive comments for this work. This work was supported
by the National Basic Research Program of China (2011CBA00400 to Z.Q., 2013CB835103
and 2009CB941302 to L.X.), CAS Hundreds of Talents Program (Z.Q.), Strategic Priority
Research Program of Chinese Academy of Sciences, Grant No.XDB02050400 (Z.Q.) and the
National Natural Science Foundation of China (U1032605 to L.X, 31100786 to R. M.).